Human cytomegalovirus (HCMV) is an opportunistic pathogen that causes diverse disease manifestations in immune deficient individuals. Intrinsic to the broad range of clinical syndromes associated with HCMV infection is the ability of the virus to infect cells of distinct and divergent developmental lineages including, epithelial, endothelial, neuronal, and blood cells. HCMV envelope glycoproteins are the initial determinants of cellular tropism and the mediators of virally-induced fusion reactions that are responsible for virus penetration and cell-to-cell spread of infection. The goal of the proposed studies is to assess the functional role of a crucial envelope glycoprotein, gB, in virus entry into host cells. Towards this end, two complementary approaches will be applied. In genetically-based studies, we will question the consequences of the loss of this abundant, key envelope component by producing a strain of virus lacking a gB protein. Production of a gB-negative virus strain is now achievable since a novel, immortalized fibroblast cell line that supports HCMV infection and plaque formation was engineered in our laboratory. We will conduct a detailed analysis of the entry properties of the wild type virus that contains a full complement of envelope proteins versus a gB- deficient virus strain that contains all envelope proteins but gB. Specific activities will be attributed to gB by virtue of the absence or inhibition of a particular event in the entry cascade with the gB-negative virus. To complement the genetic studies, biochemical characterization of a recombinant-derived, purified gB will be performed. Recovery of purified protein that retains native conformation and tertiary structure has yielded an extremely useful tool for functional studies. Using this protein, multiple criteria will be tested to further explore the existence and nature of a cellular receptor for gB. In the final stage of the grant, we will initiate structure/function analysis of gB by producing and characterizing a panel of linker-insertion mutants. Entry of HCMV into host cells is a dynamic process likely requiring the cooperative participation of multiple viral and cellular components. The realization of the proposed research goals will provide novel information not only on the functional role of gB in HCMV entry into host cells but we will also gain a greater perspective and broader comprehension of this composite and critical stage in infection of a significant human pathogen.